CD4 T-cells are thought to be a key player in protective immunity to blastomycosis and histoplasmosis, two of the principal systemic mycoses of humans and animals. However, we have recently shown, that CD8 T-cells alone, without CD4 T-cell help, can mediate efficient vaccine-induced immunity to these fungi. Resistance by CD8 T-cells was restricted by MHC class I and required the production of IFN-gamma, TNF-alpha and GM-CSF. Here, we propose to analyze the mechanisms by which these vaccines trigger and maintain long-term CD8 T cell responses to protect against experimental pulmonary blastomycosis and histoplasmosis. We will study the components of the tripartite interaction between antigen presenting cell, T-cell and antigen peptide to define the cellular and molecular requirements for priming, activation and maintenance of protective CD8 T-cells responses using in vivo and in vitro models. We postulate that memory CD8 T ceils are initiated and maintained independent of CD40/CD40L signaling, but depend on BT/CD28 co-stimulation and B-cells for survival. We also postulate that without CD4 ligation of CD40 on antigen-presenting cells yeast surface moieties induce dendritic cell (DC) maturation via Toll-like receptors (TLR); DC cross-prime naive CD8 T-cells via exogenous antigens that gain access to class I MHC unconventionally, independent of the transporter associated with antigen processing (TAP). Our aims are: 1) Elucidate factors responsible for initiation and maintenance of CD4-independent CD8 memory. We will validate that CD4 cells are indeed dispensable for long-term CD8 memory, in contrast to current dogma. We will explore the roles of CD40/CD40L and B7/CD28 signaling, and of B-cells in CD8-cell priming in the absence of CD4 help, and in the maintenance and contraction of the CD8 memory pool. Use of transgenic yeast that display an LCMV gp33 T-cell epitope will allow us to track number and function of memory CD8 cells in a way not previously possible in the fungi. 2) Define mechanisms of exogenous antigen cross-presentation to CD8 cells by DC in CD4-deficient hosts. We will explore whether and how TLR condition DC for cross-presentation of exogenous fungal antigens to induce protective CD8 T cell responses. The intracellular route by which exogenous fungal antigens access MHC class I will be studied, including the roles of TAP, degradation and loading of class I in endosomes and MHC binding on peptide regurgitation. Defining the requirements for induction and maintenance of durable memory CD8 T-cells in the absence of CD4 help, and the mechanisms of cross-presentation of exogenous fungal antigens to class I, will enhance the development of vaccine strategies against dimorphic fungi in immune compromised hosts with defective CD4 T cell immunity.